Edoardo Colombo MD
Director Flow Lab
Dept. of Pathology
Valduce Hospital
COMO , Italy
coledoco@tin.it
DETECTION OF CMV Ag ON GRANULOCYTES BY FLOW CYTOMETRY
(Edoardo Colombo MD , Carlo Colzani PhD : Valduce Hospital Como , Italy)
Note:
The Ab reacts with the CMV lower matrix protein pp65 which is the
predominant CMV antigen in leukocytes from patients with CMV infection .
(DAKO Clone AAC10)
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----------------- Leukocytes Recovering : 500 =B5L whole blood are overlaid onto 1 ml Ficoll 1.077 ; let motionless= for 20 minutes at room temperature then aspirate the supernatant ( ~ 250 =B5l= ) and wash once with PBS 0.01 M=20 -------------------------------------------------------------------------=
----------------- Cells preparation: Fix the cells with 100 =B5L CALTAG Fix&Perm Kit solution A (or Paraformaldehyde 1%) for 15 minutes at room temperature Wash with PBS 0.01 M , centrifuge and resuspend Permeabilize membranes with 100 =B5L Fix&Perm Kit solution B (or Triton-X= 100 0.1 %) for 15 minutes ; add 10 =B5L of Anti-CMV Ab (diluted 1:5 - final concentration 0.5 =B5g/ml) Wash with PBS 0.01 M, centrifuge and resuspend Transfer 100 =B5L of resuspended cells into CONTROL and TEST tubes -------------------------------------------------------------------------=
--------------------- Staining : Control : Add 10 =B5L of anti-mouse rabbit FITC antibody for 15 minutes at room temperature in dark Test: Add 10 =B5L of anti-mouse rabbit FITC antibody for 15 minutes at room temperature in dark Both: After incubation of the secondary antibody the samples are wash and resuspend in 500 =B5L PBS 0.01 M -------------------------------------------------------------------------=
---------------------- Flow Cytometry Run collecting AT LEAST 50,000 events gated by a FSC/SS bitmap ; if you a= re testing an immunodepressed patients you may find a high parasitemia , so you can stop the run early , otherwise in not-immunodepressed patients the parasitemia is very low and therefore a large number of events is needed ; if you have RAM trouble ( < 16 MB) don't save tha data as listmode. Remember that anyway the FCS 2.0 standard has problem with file greater than 30 MB (its limit is setted to 99 MB but it doesn't have a CRC for each set of data stored , so you may discover sometimes "crushed" data files...) so a 64 MB RAM is strongly raccomended. The mean of the negative is contained usually in the first decade in my four-decade histogram (I have an Elite ESP) while the positive cells are around channel 7.0 +/= - 1.5 -------------------------------------------------------------------------=
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